Protein-Carbohydrate interaction: VII. Physical and chemical studies on concanavalin A, the hemagglutinin of the jack bean

Abstract

Concanavalin A yielded a monodiperse pattern in the analytical ultracentrifuge in the pH range 2–5. The sedimentation coefficient, s o 20, w in pH 5 acetate buffer containing 0.1 m NaCl (total Γ 2 = 0.20 ) was 4S. At pH 7 and above, a two-peak schlieren pattern was observed, the faster component (about 7S) probably representing a dimer. On the basis of an s 20, w of 3.9S; a D 20, w of 5.43 × 10−7 cm 2/second, and a ī V of 0.73 ml/gm, a molecular weight of 68,000 was calculated (0.10 m acetate, NaCl, Γr 2 = 0.45 ) for the homogeneous component at pH 5. From free-boundary electrophoresis data the isoelectric point of Concanavalin A was determined to be 7.1 ± 0.1. Starch gel electrophoresis patterns of concanavalin A in borate buffer (pH 8.6) were characterized by an anodically migrating streak. Incorporation of d-glucose into the gel gave a single sharp band which migrated slowly toward the cathode, the glucose probably inhibiting protein-starch interaction. Gel filtration experiments with Biogel P-100 suggested that at pH 5 the molecular weight of Concanavalin A is in the vicinity of 50,000; at pH 7.5 concanavalin A is completely excluded, an indication that a high molecular weight species probably is formed by the association of subunits. Concanavalin A has an extinction coefficient, E 1% 1 cm of 11.4 ± 0.1, the molar ratio Mtyrosine/ Mtryptophan being 1.78. Unlike other phytohemagglutinins, concanavalin A does not contain cysteine (or cystine) or carbohydrate. Aspartic acid and serine constitute the most abundant residues, and the protein has a high amide content (68.6 groups/10 5 gm protein). Amino terminal studies showed the presence of 1.5 moles DNP-alanine/ 68,000 gm protein together with very small amounts of DNP-serine and DNP-glycine. The manganese content of concanavalin A was found to be 0.029 and 0.036%, by activation analysis and emission spectroscopy, respectively.